Anticancer agents based on prevention of protein prenylation

ABSTRACT

Prenylating enzymes are involved in modifying oncoproteins, such as RAS, so that growth of neoplastic cells becomes uncontrolled. Inactivation of such enzymes can prevent uncontrolled growth. α-Dicarbonyl compounds can be used to covalently modify and thereby inactivate prenylating enzymes such as protein farnesyltransferase and protein geranylgeranyltransferase. The compounds can be designed to enhance affinity and/or specificity for a particular protein substrate.

This application is a division of application Ser. No. 09/457,476, filedDec. 9, 1999, now U.S. Pat. No. 6,576,436, which claims benefit ofprovisional application Ser. No. 60/111,478 filed Dec. 9, 1998. Thedisclosure of the provisional application is expressly incorporated byreference herein.

TECHNICAL FIELD OF THE INVENTION

This invention relates to the use of molecules with adjacent carbonylgroups (O═C—C═O), or related functional groups, that inactivate one ormore prenyltransferases in cancer cells and thereby prevent theunrestrained division of cancer cells.

BACKGROUND OF THE INVENTION

More than half of all human cancers contain a mutant gene that encodes amutant protein known as Ras. In cancer cells, Ras activates the cells todivide in an unrestrained manner. To induce cell division, Ras must belocalized at the inner surface of the cancer cell membrane. Membranelocalization of Ras is effected by attachment of a hydrophobic group,typically the farnesyl group, which becomes attached to Ras. In somecells, the related geranylgeranyl group becomes attached instead. Bothof these groups become attached to Ras enzymatically, in a process knownas prenylation. Thus, interference with prenylation of Ras has thepotential to prevent Ras localization at the inner surface of the cancercell membrane, resulting in the cessation of unrestrained cell divisionof the cancer cell.

The enzyme that attaches the farnesyl group to Ras protein to facilitatethe latter's localization at the inner surface of the cancer cellmembrane is farnesyl protein transferase, also known as proteinfarnesyltransferase (herein referred to as FTase). The farnesyl groupbecomes attached to Ras by reaction with farnesyl diphosphate, alsoknown as farnesyl pyrophosphate (herein referred to as FPP). In otherwords, FTase catalyzes the following reaction, in which Ras becomesattached to the farnesyl group by displacement of pyrophosphate (P₂O₇⁴⁻, herein referred to as PP_(i)):

The newly formed farnesyl-Ras localizes at the inner surface of thecancer cell membrane and causes the cancer cell to divide withoutrestraint.

There is a continuing need in the art for new ways to inhibit the growthof cancer cells. There is a need in the art for new ways to inactivatetargets which are specifically involved in cancer progression anddevelopment.

SUMMARY OF INVENTION

It is an object of the invention to provide methods for inactivatingprenylating enzymes.

It is another object of the invention to provide methods for screeningtest compounds for the ability to inactivate prenylating enzymes.

It is yet another object of the invention to provide methods forinhibiting the growth of a cancer cell.

It is still another object to provide pharmaceutical compositions fortreating cancer.

It is another object to provide new compounds useful for treatingcancers or inhibiting enzymes.

These and other objects of the invention are provided by one or more ofthe embodiments described below. In one embodiment a method ofinactivating a prenylation enzyme is provided. The method comprises thestep of contacting a prenylation enzyme with an α-dicarbonyl compoundhaving formula (I): R1-(C═O)—(C═O)—R2, wherein R1 is selected from thegroup consisting of hydrogen, alkyl, alkenyl, alkynyl, and aryl; andwherein R2 is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, and aryl; and whereby the activity of the prenylationenzyme is reduced by at least 50%.

According to another embodiment of the invention, a method of screeningcompounds as anti-tumor agents is provided. The method comprises thestep of contacting a test compound with a prenylation enzyme. The testcompound has formula (I): R1-(C═O)—(C═O)—R2, wherein R1 is selected fromthe group consisting of hydrogen, alkyl, alkenyl, alkynyl, and aryl; andR2 is selected from the group consisting of hydrogen, alkyl, alkenyl,alkynyl, and aryl. Prenylation activity of the enzyme is measured. Atest compound which reduces prenylation activity at least 50% isidentified as a candidate anti-tumor agent.

According to another aspect of the invention a method of inactivating aprenylation enzyme is provided. The method comprises the step ofcontacting a prenylation enzyme with an α-dicarbonyl compound havingformula (II): R1-(C═O)—(C═O)—L—(C═O)—(C═O)—R2, wherein R1, R2, and L areindependently selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, and aryl. The activity of the prenylation enzyme isreduced by at least 50%.

According to another aspect of the invention a method of screeningcompounds as anti-tumor agents is provided. A test compound is contactedwith a prenylation enzyme. The test compound has formula (II):R1-(C═O)—(C═O)—L—(C═O)—(C═O)—R2, wherein R1, R2, and L are independentlyselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,and aryl. Prenylation activity of the enzyme is measured. A testcompound which reduces prenylation activity at least 50% is a candidateanti-tumor agent.

Another embodiment of the invention provides yet another method ofinactivating a prenylation enzyme. A prenylation enzyme is contactedwith an α-dicarbonyl compound having formula (III): A-L-D-R, wherein Ais selected from the group consisting of an anionic group, a group thatcan spontaneously become anionic at physiological pH, and a group thatcan enzymatically become anionic at physiological pH; wherein L is alinker group; wherein D is a dicarbonyl functional group or a maskedform of said group, and wherein R is selected from the group consistingof hydrogen, alkyl, alkenyl, alkynyl, and aryl. The activity of theprenylation enzyme is reduced by at least 50%.

Still another aspect of the invention is a method of screening compoundsas anti-tumor agents. A test compound is contacted with a prenylationenzyme. The test compound has formula (III): A-L-D-R, wherein A isselected from the group consisting of an anionic group, a group that canspontaneously become anionic at physiological pH, and a group that canenzymatically become anionic at physiological pH; wherein L is a linkergroup; R is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, and aryl, and wherein D is a dicarbonyl functionalgroup or a masked form of said group. Activity of the prenylation enzymeis measured. A test compound which reduces prenylation activity at least50% is a candidate anti-tumor agent.

Also provided as an embodiment of the invention is a method ofinhibiting growth of cancer cells. A cancer cell is contacted with anα-dicarbonyl compound having a formula: R1-(C═O)—(C═O)—R2. R1 isselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,and aryl. R2 is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, and aryl. The growth of the cancer cell isconsequently inhibited.

Another aspect of the invention is a method of inhibiting the growth ofa cancer cell in which a cancer cell is contacted with an α-dicarbonylcompound having a formula: R1-(C═O)—(C═O)—L—(C═O)—(C═O)—R2. R1, L, andR2 are independently selected from the group consisting of hydrogen,alkyl, alkenyl, alkynyl, and aryl. The growth of the cancer cell isconsequently inhibited.

Another embodiment of the invention is a method of inhibiting the growthof a cancer cell. A cancer cell is contacted with an α-dicarbonylcompound having a formula: A-L-D-R. A is selected from the groupconsisting of an anionic group, a group that can spontaneously becomeanionic at physiological pH, and a group that can enzymatically becomeanionic at physiological pH. L is a linker group, and R is selected fromthe group consisting of hydrogen, alkyl, alkenyl, alkynyl, and aryl. Dis a dicarbonyl functional group or a masked form of said group. Thegrowth of the cancer cell is consequently inhibited.

Also provided by the present invention is a pharmaceutically acceptableformulation. The formulation comprises a compound according to FormulaI, Formula II, or Formula III, and a pharmaceutically acceptableexcipient.

Additionaly provided by the present invention is a compound according toFormula I, Formula II, or Formula III. The compound is notphenylglyoxal, biphenyldiglyoxaldehyde, 2-oxododecanal, or2,3-pentanedione.

The present invention thus provides a method for hampering or preventingthe proliferation of cancer cells, resulting in a decrease in tumor sizeand/or disappearance of the cancer. It acts by interference with cancercell biochemistry, in which the enzyme farnesyl protein transferase,geranylgeranyl protein transferase, and/or another prenylation enzymeacts on oncogenic proteins, such as RAS, or other growth-relatedcellular protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Assay of FTase after incubation of the enzyme in the absence(upper curve) and presence (lower curve) of inactivator (10 mM PGO for10 min). Activity of human recombinant FTase was monitored by theincrease in fluorescence at λ=505 nm, with farnesyl pyrophosphate andDs-GCVLS (as a substitute for Ras) as substrates, after removal of PGOwith a high molecular weight cutoff microconcentrator. The lower curveshows the virtual absence of enzyme activity following preincubation ofFTase with PGO. Each point is the average of duplicate determinations.

FIG. 2. Kinetics of inactivation of FTase by PGO. The rate ofinactivation of FTase is dependent on PGO concentration. Loss of enzymeactivity is faster at higher concentrations of PGO: (▪) PGO absent, (♦)5 mM PGO, and (Δ) 10 mM PGO. At 20 mM PGO, enzyme activity was lost in 2min of incubation (data not shown). Assays were carried out afterremoval of PGO with a high molecular weight cutoff microconcentrator.Each point is the average of duplicate determinations.

FIG. 3. Farnesyl pyrophosphate protection of FTase from inactivation byPGO. Points are determined by 100×(FTase activity after incubation withFPP and PGO)/(FTase activity after incubation with FPP without PGO).Assays were carried out after removal of FPP and PGO with a highmolecular weight cutoff microconcentrator. Each point is the average ofduplicate determinations.

FIG. 4. Kinetics of inactivation of FTase by 2-oxododecanal. The rate ofinactivation of FTase is dependent on 2-oxododecanal concentration. Lossof enzyme activity is faster at higher concentrations of 2-oxododecanal:(▪) 2-oxododecanal absent, (♦) 5 mM 2-oxododecanal, and (Δ) 10 mM2-oxododecanal. At 35 mM 2-oxododecanal, enzyme activity was lost in 2min of incubation (data not shown). Dimethyl sulfoxide was present at 5%in the incubation mixtures (including the control). Assays were carriedout after removal of 2-oxododecanal with a high molecular weight cutoffmicroconcentrator. Each point is the average of duplicatedeterminations.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Key targets in a strategy to retard cancer cell proliferation are theprenylation enzymes, such as the enzyme FTase. By reducing or destroyingFTase activity, we have found that we can prevent Ras farnesylation,which in turn prevents localization of Ras at the inner surface of thecancer cell membrane, thereby preventing Ras from causing the cancercells to divide and proliferate.

Many substances are known to block FTase activity and preventfarnesylation of Ras. They consist of inhibitors of the enzyme FTase,and they generally operate by blocking the binding of Ras or FPP (orboth) to FTase. Without the normal substrates (Ras and FPP) being ableto bind to FTase, FTase cannot transfer the farnesyl group from FPP toRas. Inhibitors structurally mimic one or both of the natural substratesof the enzyme, Ras and/or FPP. Their binding is noncovalent andreversible. In other words, the binding of the inhibitor to FTase doesnot involve the formation of covalent bonds between the inhibitor andFTase. Instead, hydrophobic forces, hydrogen bonding, electrostaticattraction, etc. are the forces principally responsible for binding ofthe inhibitor to the enzyme FTase. These binding forces allow theinhibitor to block the site on FTase where the normal substrates need tobind for farnesylation of Ras to take place.

We have found that a potentially more useful method of preventing FTasefrom farnesylating Ras can be achieved, namely inactivation of FTase.Interaction of FTase with substances that are inactivators results inthe covalent attachment of a part or all of the inactivator to FTase. Inprinciple, the covalent attachment can be irreversible or nearlyirreversible. Also, the specificity of the inhibitor for the targetenzyme FTase can be more finely tuned because it is based on twoparameters: (1) structural similarity to the normal substrates (FPPand/or Ras); (2) a reactivity that is appropriate for the chemicalgroups that are a part of the enzyme FTase, particularly those thatparticipate in the actual process catalyzed by FTase (i.e., those thatmore- or-less directly participate in the transfer of the farnesyl groupof FPP to Ras).

The fact that FPP loses pyrophosphate, which is anionic, might beindicative of the presence of a structural feature in FTase that wouldassist in the displacement of pyrophosphate, for example by ionicbonding to a positively charged group in the enzyme's active site. Onesuch possibility is the side chain of arginine [R—NH═C(NH₂)₂ ⁺]. Indeed,the X-ray crystallographic structure determination of FTase reveals thepresence of arginine residues in the vicinity of the hypotheticalbinding site of FPP, which further suggests the possible role ofarginine in assisting with the removal of pyrophosphate as the farnesylgroup becomes bonded to Ras.

Some chemical reagents that react with the side chain of arginineresidues in proteins possess a functional group that comprises at leasttwo adjacent carbonyl groups, i.e., O═C—C═O. Such a functional groupbinds covalently to the nitrogens of the side chain of arginine, causingthe vicinity of the arginine to become blocked and possibly the positivecharge to be lost. Hypothetical structures of the adduct(s) of thearginine side chain and the dicarbonyl compound phenylglyoxal are shownbelow:

Our invention consists of chemical agents bearing at least two adjacentcarbonyl groups (O═C—C═O) designed to react (i.e., covalently interact)with the arginine side chain. Furthermore, to target better thesubstance to FTase in preference to other enzymes, and to target betterthe substance to the active site of FTase in preference to the externalor noncatalytic portion of the enzyme, a preferred embodiment of theinvention consists of a substance with at least one dicarbonylfunctional group and other structural features which favor the bindingof the agent to the active site of FTase. For example, the active siteof FTase is hydrophobic to bind the natural substrate FPP which containsa hydrophobic region (i.e., the farnesyl group). A hydrophobic substancethat also contains the dicarbonyl functional group is preferred, a classrepresented by the following generalized structural formula:

wherein R and R′ represent hydrogen and/or a hydrophobic group, such asan alkyl, alkenyl, alkynyl, and aryl group. The group can be cyclic ornoncyclic, branched or unbranched, with or without unsaturation (e.g.,aryl, alkenyl) or substituents. Preferably the group is designed toimpart specificity of the substance for binding to and/or inactivationof FTase.

The term “alkyl,” as used alone or in combination herein, refers to anunsubstituted or optionally substituted, straight, or branched chainsaturated hydrocarbon group containing from one to twenty-five carbonatoms, preferably from one to fifteen carbons, such as methyl, ethyl,n-propyl, n-butyl, pentyl, hexyl, heptyl, octyl, the various branchchain isomers thereof, such as isopropyl, isobutyl, sec-butyl,tert-butyl, isohexyl and the like. The alkyl group may be optionallysubstituted by one or more substituents, and generally no more thanthree, and most often just one substituent. Preferred optionalsubstituents include halo, alkoxy, amino, mono- and di-substitutedamino, aryl, carboxylic acid, heterocyclo, heteroaryl, cycloalkyl,hydroxy, trifluoromethoxy and the like. The term “lower alkyl” refers tosuch alkyl groups containing from one to five carbon atoms.

The term “alkoxy,” as used alone or in combination herein, refers to analkyl group, as defined above, covalently bonded to the parent moleculethrough an —O— linkage, such as methoxy, ethoxy, propoxy, isopropoxy,butoxy, t-butoxy and the like.

The term “alkoxyalkyl” refers specifically to an alkyl group substitutedwith an alkoxy group.

The term “aryloxy,” as used alone or in combination herein, refers to anaryl group, as defined below, covalently bonded to the parent moleculethrough an —O— linkage. An example of an aryloxy is phenoxy.

The term “cycloalkoxy,” as used alone or in combination herein, refersto a cycloalkyl group, as defined below, covalently bonded to the parentmolecule through an —O— linkage.

The term “alkylthio,” as used alone or in combination herein, refers toan alkyl group, as defined above, covalently bonded to the parentmolecule through an —S— linkage.

The term “alkenyl,” as used alone or in combination herein, refers to analkyl group, as defined above, containing one or more carbon-carbondouble bonds, preferably one or two double bonds. Examples of alkenylinclude ethenyl, propenyl, 1,3-butadienyl, and 1,3,5-hexatrienyl.

The term “alkynyl,” as used alone or in combination herein, refers to analkyl group, as defined above, containing one or more carbon-carbontriple bonds, preferably one or two triple bonds.

The term “cycloalkyl,” as used alone or in combination herein, refers toan unsubstituted or optionally substituted, saturated cyclic hydrocarbongroup containing three to eight carbon atoms. The cycloalkyl group mayoptionally be substituted by one or more substituents, and generally nomore than three, and most often just one substituent. Preferred optionalsubstituents include alkyl, halo, amino, mono- and di-substituted amino,aryl, hydroxy and the like.

The term “haloalkyl” is a species of alkyl as defined herein, andparticularly refers to an alkyl, preferably a lower alkyl, substitutedwith one or more halogen atoms, and preferably is a C₁ to C₄ alkylsubstituted with one to three halogen atoms. One example of a haloalkylis trifluoromethyl.

The term “alkanoyl” as used alone or in combination herein refers to anacyl radical derived from an alkanecarboxylic acid (alkyl-C(O)—),particularly a lower alkanecarboxylic acid, and includes such examplesas acetyl, propionyl, butyryl, valeryl, and 4-methylvaleryl.

The term “aroyl” means an acyl radical derived from an aromaticcarboxylic acid, such as optionally substituted benzoic or naphthoicacids and specifically including benzoyl and 1-naphthoyl.

The term “aminocarbonyl” means an amino-substituted carbonyl (carbamoylor carboxamide) wherein the amino group is a primary amino (—NH₂).Substituted aminocarbonyl refers to secondary (mono-substituted amino)or tertiary amino (di-substituted amino) group, as defined below,preferably having as a substituent(s) a lower alkyl group.

The term “aminoalkanoyl” means an amino-substituted alkanoyl wherein theamino group is a primary amino group (-alkyl-C(O)—NH₂). The term“substituted aminoalkanoyl” refers to related secondary(mono-substituted amino) or tertiary amino (di-substituted amino) group,as defined below.

The term “carbocycloalkyl” when used alone or in combination refers toan unsubstituted or optionally substituted, stable, saturated orpartially unsaturated monocyclic, bridged monocyclic, bicyclic, andspiro ring carbocycles of 3 to 15 carbon atoms such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclohexyl,bicyclooctyl, bicyclononyl, spirononyl and spirodecyl. Cycloalkyls arethus one specific subset of carbocycloalkyls. The term “optionallysubstituted” as it refers to “carbocycloalkyl” herein indicates that thecarbocycloalkyl group may be substituted at one or more substitutablering positions by one or more groups independently selected from alkyl(preferably lower alkyl), alkoxy (preferably lower alkoxy), nitro,monoalkylamino (preferably a lower alkylamino), dialkylamino (preferablya di[lower]alkylamino), cyano, halo, haloalkyl (preferablytrifluoromethyl), alkanoyl, aminocarbonyl, monoalkylaminocarbonyl,dialkylaminocarbonyl, alkylamido (preferably lower alkylamido),alkoxyalkyl (preferably a lower alkoxy[lower]alkyl), alkoxycarbonyl(preferably a lower alkoxycarbonyl), alkylcarbonyloxy (preferably alower alkylcarbonyloxy) and aryl (preferably phenyl), said aryl beingoptionally substituted by halo, lower alkyl and lower alkoxy groups.Generally, there is no more than one optional substituent.

The term “heterocyclo” as used, alone or in combination, herein refersto an unsubstituted or optionally substituted, stable, saturated, orpartially unsaturated, monocyclic, bridged monocyclic, bicyclic, andspiro ring system containing carbon atoms and other atoms selected fromnitrogen, sulfur and/or oxygen. Preferably, a heterocyclo group is a 5or 6-membered monocyclic ring or an 8–11 membered bicyclic ring whichconsists of carbon atoms and contains one, two, or three heteroatomsselected from nitrogen, oxygen and/or sulfur. Heterocyclo includesbenz-fused monocyclic cycloalkyl groups having at least one suchheteroatom. The term “optionally substituted” as it refers to“heterocyclo” herein indicates that the heterocyclo group may besubstituted at one or more substitutable ring positions by one or moregroups independently selected from alkyl (preferably lower alkyl andincluding haloalkyl (preferably trifluoromethyl)), alkoxy (preferablylower alkoxy), nitro, monoalkylamino (preferably a lower alkylamino),dialkylamino (preferably a di[lower]alkylamino), cyano, halo, alkanoyl,aminocarbonyl, monoalkylaminocarbonyl, dialkylaminocarbonyl, alkylamido(preferably lower alkylamido), alkoxyalkyl (preferably a loweralkoxy[lower]alkyl), alkoxycarbonyl (preferably a lower alkoxycarbonyl),alkylcarbonyloxy (preferably a lower alkylcarbonyloxy) and aryl(preferably phenyl), said aryl being optionally substituted by halo,lower alkyl and lower alkoxy groups. Generally, there is no more thanone optional substituent. Several non-limiting examples of suchheterocyclo groups are illustrated below:

The heterocyclo group may be, and generally is attached to the parentstructure through a carbon atom, or alternatively may be attachedthrough any heteroatom of the heterocyclo group that results in a stablestructure.

The term “heteroaryl” as used alone or in combination, herein refers toan unsubstituted or optionally substituted, stable, aromatic monocyclicor bicyclic ring system containing carbon atoms and other atoms selectedfrom nitrogen, sulfur and/or oxygen. Preferably, a heteroaryl group is a5- or 6-membered monocyclic ring (optionally benzofused) or an 8–11membered bicyclic ring which consists of carbon atoms and contains one,two, or three heteroatoms selected from nitrogen, oxygen and/or sulfur.The term “optionally substituted” as it refers to “heteroaryl” hereinindicates that the heteroaryl group may be substituted at one or moresubstitutable ring positions by one or more groups independentlyselected from alkyl (preferably lower alkyl and including haloalkyl(preferably trifluoromethyl)), alkoxy (preferably lower alkoxy), nitro,monoalkylamino (preferably a lower alkylamino), dialkylamino (preferablya di[lower]alkylamino, cyano, halo, alkanoyl, aminocarbonyl,monoalkylaminocarbonyl, dialkylaminocarbonyl, alkylamido (preferablylower alkylamido), alkoxyalkyl (preferably a lower alkoxy[lower]alkyl),alkoxycarbonyl (preferably a lower alkoxycarbonyl), alkylcarbonyloxy(preferably a lower alkylcarbonyloxy) and aryl (preferably phenyl), saidaryl being optionally substituted by halo, lower alkyl and lower alkoxygroups. Generally, there is no more than one optional substituent.Several non-limiting examples of such heteroaryl groups are illustratedbelow:

The heteroaryl group may be, and generally is attached to the parentstructure through a carbon atom or alternatively may be attached throughany heteroatom of the heteroaryl group that results in a stablestructure. In the foregoing structures it also is contemplated that anitrogen could be replaced with an N-oxide.

Both heterocyclo and heteroaryl also are intended to embrace benzo fusedstructures such as 1,2-methylenedioxybenzene and 1,4-benzodioxan.

The terms “halo” and “halogen” as used herein to identify substituentmoieties, represent fluorine, chlorine, bromine or iodine, preferablychlorine or fluorine.

The term “aryl,” when used alone or in combination, refers to anunsubstituted or optionally substituted monocyclic or bicyclic aromatichydrocarbon ring system having 6 to 12 ring carbon atoms. Preferred areoptionally substituted phenyl, 1-naphthyl, or 2-naphthyl groups. Thearyl group may optionally be substituted at one or more substitutablering positions (generally at no more than three positions and most oftenat one or two positions) by one or more groups independently selectedfrom alkyl (including haloalkyl (preferably trifluoromethyl anddifluoromethyl)), alkenyl, alkynyl, alkoxy, aryloxy, nitro, hydroxy,amino, mono- and di-substituted amino, cyano, halo, alkanoyl,aminocarbonyl, carboxylic acid, carboxylic acid esters, carboxylic acidamide, an optionally substituted phenyl (optionally substituted by halo,lower alkyl and lower alkoxy groups), heterocyclo, or heteroaryl.Preferably, the aryl group is phenyl optionally substituted with up tofour and more usually with one or two groups, preferably selected fromlower alkyl, lower alkoxy, as well as cyano, trifluoromethyl and halo.

The terms “aralkyl” and “(aryl)alkyl,” alone or in combination are aspecies of alkyl as defined herein, and particularly refers to an alkylgroup as defined above in which one hydrogen atom is replaced by an arylgroup as defined above, and includes benzyl, and 2-phenylethyl.

The terms “(heterocyclo)alkyl” and “(heteroaryl)alkyl” alone or incombination can be considered a species of alkyl as defined herein, andparticularly refers to an to an alkyl group as defined above in whichone hydrogen atom is replaced by a heterocyclo group as defined above,or by a heteroaryl group as defined above.

The term “alkoxycarbonyl” alone or in combination means a radical of theformula —C(O)-alkoxy, in which alkoxy is as defined above.

The term “alkylcarbonyloxy” alone or in combination means a radical ofthe formula —O—C(O)-alkyl, in which alkyl is as defined above.

The term “alkoxyalkanoyl” alone or in combination means a radical of theformula --alkyl-C(O)—O-alkyl.

The term “carboxyalkyl” alone or in combination means a radical of theformula --alkyl-C(O)—OH.

The term “substituted amino” embraces both mono and di-substitutedamino. These terms, alone, or in combination, mean a radical of theformula —NR′R″, where, in the case of mono-substitution, one of R′ andR″ is a hydrogen and the other is selected from alkyl, cycloalkyl, aryl,heterocyclo, (aryl)alkyl, (heterocyclo)alkyl, heteroaryl andhetero(aryl)alkyl; in the case of di-substitution, R′ and R″ areindependently selected from alkyl, cycloalkyl, aryl, heterocyclo, andheteroaryl, or R′ and R″ together with the nitrogen atom to which theyare both attached form a three to eight-membered heterocyclo orheteroaryl radical.

The term “amido” refers to the group (—NH—) and the term “substitutedamido” embraces a radical of the formula (—NR—) where R′ has the meaningabove in connection with substituted amino.

The terms “alkanoylamido,” “aroylamido,” “heterocyclocarbonylamido” and“heteroaroylamido” mean groups of the formula R—C(O)—NH— where R is analkyl, aryl, heteroaryl or heterocyclo group.

The terms “heteroaroyl” and “heterocyclocarbonyl” when used alone or incombination means groups of the formula R—C(O)—— where R is a heteroarylor heterocyclo group.

Unless otherwise defined, the term “optionally substituted” as usedherein, refers to the substitution of a ring system at one or morepositions with one or more groups selected from: C₁₋₅ alkyl, C₁₋₅alkoxy, an optionally substituted phenyl, cyano, halo, trifluoromethyl,C₁₋₅ alkoxycarbonyl, C₁₋₅ alkyl carbonyloxy, mono- and bis-(C₁₋₅alkyl)-carboxamide, C₁₋₅ alkylamido, nitro, and mono- and bis-(C₁₋₅alkyl)-amino.

Applicants recognize that there may be some overlap in some of thedefinitions of the various radical groups. Specific groups arementioned, however, such as (aryl)alkyl, and may be particularlyidentified in the claims, in order to emphasize their positive inclusionin the described subject matter, as not only an optional substituent.

As used herein, when a particular radical generally understood to have asingle point of attachment to a core structure, such as an alkyl group,is identified in connection with a structure that must have two pointsof attachment in the structural core (such as with the element L informula (II)), it is understood that the named radical, e.g., alkyl,refers to the parent radical with a hydrogen or a site of unsaturationremoved to create the second point of attachment so as to provide therequired structure.

Anionic groups according to the present invention, designated as “A”herein, include carboxylate, sulfate, sulfonate, sulfinate,sulfonamides, sulfones, phosphate, phosphonate, phosphinate, tetrazoles,thiophosphate, pyrophosphate, enolate, or a precursor group that can beconverted to an anionic group, either spontaneously or enzymatically incells.

A specific embodiment of this invention is phenylglyoxal, shown below,although many other compounds that can achieve the desired effect arereadily apparent to those skilled in the art, having regard for thisdisclosure:

-   -   phenylglyoxal

Two additional categories of specific embodiments, in which an aliphaticchain is capable of binding to the active site of FTase by hydrophobicinteractions, are defined as follows:

Also, substances with complex substituents might be better mimics of thefarnesyl group of the substrate FPP and function more effectively asinhibitors, as for example, the compounds below:

An additional compound that consists of the general formulaR—(C═O)—(C═O)—R′, or a masked form of such a group as denoted elsewherein this application (e.g. acetal, ketal, etc.), and is contemplated bythis disclosure is the following:

-   -   5,9-dimethyl-8-decene-2,3-dione

The synthesis of the above compound may be carried out as follows:

An additional compound that consists of the general formulaR—(C═O)—(C═O)—R′, or a masked form of such a group as denoted elsewherein this application (e.g. acetal, ketal, etc.), and is contemplated bythis disclosure is the following:

The synthesis of the above compound may be carried out according to thefollowing scheme:

A further example of the class of compounds of the typeR—(C═O)—(C═O)—R′, or a masked form of such a group as denoted elsewherein this application (e.g. acetal, ketal, etc.), is exemplified bydehydroascorbic acid 6-palmitate, shown below:

This compound may advantageously be synthesized by oxidation of ascorbicacid 6-palmitate, as follows:

Other embodiments contemplated include compounds in which the—(CH₂)₁₄CH₃ group shown in the structural formula of the 6-palmitate isreplaced by a group R as defined elsewhere herein. These include other6-esters of dehydroascorbic acid, such as dehydroascorbic acid6-farnesenoate, in which the —(CH₂)₁₄CH₃ group shown in the structuralformula of the 6-palmitate is replaced by the—CH═C(CH₃)CH₂CH₂CH═C(CH₃)CH₂CH₂CH═C(CH₃)₂ group. Dehydroascorbic acidesters contemplated by this disclosure are inclusive of the 5-isomers aswell as the 6-isomers.

Some cancer cells in which farnesylation of Ras is blocked employ therelated prenylation reaction geranylgeranylation to attach a hydrophobicgroup to Ras to accomplish membrane localization and continued cancerousbehavior of the cell. The enzyme that attaches the geranylgeranyl groupto Ras protein to facilitate the latter's localization at the innersurface of the cancer cell membrane is geranylgeranyl proteintransferase, also known as protein geranylgeranyltransferase (hereinreferred to as GGTase). The geranylgeranyl group becomes attached to Rasby reaction with geranylgeranyl diphosphate, also known asgeranylgeranyl pyrophosphate (herein referred to as GGPP). In otherwords, GGTase catalyzes the following reaction, in which Ras becomesattached to the geranylgeranyl group by displacement of pyrophosphate(PP_(i)):

The newly formed geranylgeranyl-Ras localizes at the inner surface ofthe cancer cell membrane and causes the cancer cell to divide withoutrestraint. Thus, a key target in a strategy to retard cancer cellproliferation is the enzyme GGTase. By reducing or destroying GGTaseactivity, either in combination with inactivation of Ras farnesylationor independently, we hope to prevent Ras geranylgeranylation, which inturn should prevent localization of Ras at the inner surface of thecancer cell membrane, thereby preventing Ras from causing the cancercells to divide and proliferate.

Geranylgeranylation of Ras results in the loss from GGPP ofpyrophosphate, which is anionic, a process that might be facilitated bya chemical group in GGTase's active site that would favorably interactwith pyrophosphate, such as by ionic bonding to a positively chargedgroup like the side chain of arginine [R—NH═C(NH₂)₂ ⁺]. Thus, chemicalreagents that react with the arginine side chain, such as those thatpossess the functional group consisting of two adjacent carbonyl groups,i.e., O═CC═O, might effectively prevent one or more arginine residues inGGTase from assisting in the pyrophosphate departure, thereby blockingor impeding geranylgeranylation of Ras. Furthermore, to target betterthe substance to GGTase in preference to other enzymes, and to targetbetter the substance to the active site of GGTase in preference to theexternal or noncatalytic portion of the enzyme, a preferred embodimentof the invention would consist of a substance with the dicarbonylfunctional group and other structural features that would direct orotherwise favor the binding of the agent to the active site of GGTase.

To fine-tune the inactivator to fit the active site of FTase or GGTase,variation of the distance between the arginine-binding end and thefarnesyl-mimicking or geranylgeranyl-mimicking end can be achievedthrough alteration of the length of the spacer, i.e., by variation of“n” from 0 to 10 in the generalized diagram below:

Additional inactivators incorporating aromatic groups for enhancedbinding to the hydrophobic binding site of FTase or GGTase areexemplified by the following compound, although there are numerouspossible variants of this type of compound (e.g., the location of thearomatic ring and the pattern of substitution as ortho, meta, or para; aheterocyclic ring; a nonaromatic ring; multiple rings; etc.):

A further embodiment of this invention is based on the observation thatthere are two arginine residues in the active site of FTase. Becausesuch arginine residues are at a characteristic distance unique to FTase,selective reaction with FTase over other enzymes and proteins can beaccomplished by use of a substance in which two dicarbonylfunctionalities are separated by a spacer, such substances beingrepresented by the following generalized formula:D-L-Din which D is a dicarbonyl functional group, O═C—C═O, or a masked formof such a group as denoted elsewhere in this application (e.g. acetal,ketal, etc.), and L is a linker group that is compatible with and/or hasan affinity for the prenylation enzyme. One embodiment of thisinvention, in which two dicarbonyl functional groups are connected via alinker, is 4,4′-biphenyldiglyoxaldehyde, shown below:

This category of compounds is further exemplified by thebis(α-oxoaldehyde) below:

The spacer, (CH₂)_(n), can be varied in length and in othercharacteristics (e.g., steric bulk, etc.) to optimize the selectivityfor FTase and the ability to block the FTase active site. Alternativesto the bis(α-oxoaldehyde) above include substances of thebis(α-oxoketone) type or compounds with both an α-oxoketone andα-oxoaldehyde functionality, as well as others readily apparent to thoseskilled in the art, having regard for this disclosure. Thus, reaction ofFTase with compounds such as the bis(dicarbonyl) compound shown abovemight occur with high efficiency and selectivity, and the covalentattachment of such compounds to FTase might be particularly effective atblocking the active site from utilizing the natural substrates FPP andRas protein, which need to bind to FTase in the vicinity of the twoarginine residues.

Another embodiment of the invention utilizes the characteristics of theC-terminus of the Ras protein, i.e., the negative charge, fornoncovalent binding to one of the arginine residues and the dicarbonylfunctionality for covalent binding to the other arginine residue. Thetheory has been put forth that one of the arginine residues holds theRas protein in the active site through electrostatic binding of thearginine residue's positive charge to the carboxyl terminus of Rasprotein, which is negatively charged. The other arginine residue bindsFPP through the electrostatic attraction of the negative charge of FPPto the positive charge of the arginine side chain. Thus, one such classof compounds that interfere with protein prenylation that iscontemplated by this disclosure consists of four-part moleculessymbolized as follows:A-L-D-Rin which each part is defined as follows:

A is (1) an anionic group, e.g., carboxylate, sulfate, sulfonate,sulfinate, phosphate, phosphonate, thiophosphate, pyrophosphate,enolate, sulfonamide, sulfone, or tetrazole, (2) a group that can becomeanionic at physiological pH, e.g. CO₂H by deprotonation, or (3) a groupthat can be acted upon by an enzyme to reveal a group that can becomeanionic at physiological pH, e.g. the ester R′CO₂R is hydrolyzed byendogenous esterases to form the acid R′CO₂H, which subsequently ionizesat physiological pH to R′CO₂ ⁻;

L is a linker group that is compatible with and/or has an affinity forthe prenylation enzyme, for example an alkyl, alkenyl, alkynyl, or arylgroup.

D is a dicarbonyl functional group (O═C—C═O) or a masked form of such agroup as denoted elsewhere in this application (e.g., acetal, ketal,etc.).

R is a group selected from the group consisting of an alkyl, alkenyl,alkynyl, or aryl group.

Specific embodiments of such a four-part molecule are the followingcamphorquinone sulfonamides:

in which n is advantageously equal to 4, 5, 6, or 7, or another valuefor optimal binding to prenyltransferases. It will be apparent to thoseskilled in the art and having regard for this disclosure that otherlinking groups L, other anion-generating or anionic groups A, and otherdicarbonyl groups D, or masked forms of such groups as denoted elsewherein this application (e.g. acetals, ketals, etc.), can be advantageouslysubstituted for the polymethylene chain —(CH₂)_(n)—, carboxyl group, andcamphorquinone moiety, respectively, in the above structure. Forexample, a prodrug, i.e., a substance that has advantages of stability,solubility, cell permeability, etc., but that liberates the active or amore active substance in the living tissues, e.g. spontaneously and/orby action of endogenous enzymes, light, and/or heat, couldadvantageously be formulated to be the camphorquinone sulfonamide estersrepresented by the structure shown below:

in which R may be any of a very wide variety of groups, such as simplestraight-chain, branched, or cyclic and heterocyclic aliphatic groups(including functional groups containing heteroatoms, such as etherlinkages, amino and hydroxyl groups), and aromatic groups, includingheterocycles.

Both of the enantiomeric camphorquinone sulfonamides (i.e., both (+) and(−) stereoisomers) are contemplated by this patent and might beadvantageously used for interference with protein prenylation.

The synthesis of camphorquinone sulfonamides can be carried out asillustrated below:

Additionally, the compound below, when in its ionized form (thepredominant form present at physiological pH), has the characteristicsrequired for the noncovalent binding of the carboxylate terminus to onearginine residue, and the covalent binding of the dicarbonylfunctionality to the other arginine residue:(COOH)—(CH₂)_(m)—(C═O)—(COOH)⇄(COO⁻)—(CH₂)_(m)—(C═O)—(COOH)+H⁺When m=9, the compound is 11,12-dioxododecanoic acid, a preferredcompound of the invention. Typically m=1 to 25, more preferably 5–13.

Other embodiments can combine the carboxylic acid group and anα-oxoaldehyde or, alternatively, a carboxylic acid group and anα-oxoketone group within the same molecule, or other combinations thatare readily apparent to those skilled in the art, having regard for thisdisclosure. By modification of the linker group's structure (e.g.,length, steric bulk, etc.), the specificity of the compound for FTaseand the effectiveness of the compound against FTase can be fine-tuned.Use of known non-covalent inhibitors of FTase and GGTase as linker or Rgroups is preferred to increase specificity for the enzyme active site.

For example, the tetrapeptide portion of Ras that is recognized by FTase(referred to as CAAX, in which C represents cysteine, A represents analiphatic amino acid, and X represents a limited subset of the naturallyoccurring amino acids, of which serine is an example) can be utilized incombination with a dicarbonyl functionality to target the inactivator toits target, FTase. Such a compound might be a useful inactivator. Itmight, however, be hydrolyzed by cellular proteases, so a peptidomimeticanalogue less prone to cellular hydrolysis might show superior lifetimein a cell, thereby enhancing its chances of combining with FTase andinactivating it. An embodiment of a peptidomimetic analogue of thetetrapeptide in which a dicarbonyl functionality is present is shownbelow:

In the structure above, R₁ is a substituent suitable for recognition andbinding to FTase, and R₂ is typically H or CH₃, for reaction with anarginine residue in the FTase active site. Many other embodiments ofthis type are readily apparent to those skilled in the art, havingregard for this disclosure.

There are numerous ways to synthesize α-dicarbonyl compounds. One is byoxidation of a monocarbonyl compound with selenium dioxide (SeO₂), shownbelow:

Another method of preparation of a-dicarbonyl compounds is by alkylationof 2,3-butanedione, illustrated below:

Another means of synthesis of α-dicarbonyl compounds is illustratedbelow, starting with citronellal:

Other embodiments of the invention are substances in which one or morecarbonyl groups are masked as a hydrate [C(OH)₂], a hemiacetal orhemiketal [C(OH)(OR″)], an acetal or a ketal [C(OR″)(OR′″)], an acylalor related compound [C(OC(═O)R″)(OC(═O)R′″)], a bisulfite additioncompound [C(OH)(SO₃ ⁻)], an enol (C═COH), an enol ether (C═COR″), anenol ester [C═COC(═O)R″], and so forth, wherein R″ and R′″, which may bethe same or different, are alkyl, alkenyl, and/or aryl groups. Suchmasked carbonyl groups may produce the carbonyl form of the reagent insolution. Acetals and ketals with hydrophobic side chains, includingthose that resemble the farnesyl group, are shown below:

The examples below show that FTase is sensitive to inactivation byphenylglyoxal and by 2-oxododecanal, both of which are a-dicarbonylcompounds. New opportunities exist for the development of -dicarbonylcompounds and related substances that might prove to be inactivators ofprenylation enzymes with varying potencies and specificities. A specificprotein designated K-Ras is the most common mutant Ras protein found inhuman cancers. Farnesylation inhibitors, however, cannot preventcellular prenylation of K-Ras because geranylgeranylation of K-Ras takesplace. If GGTase has an active-site arginine, -dicarbonyl compounds likePGO might likewise be effective inactivators of that enzyme as well.

The ability of the α-dicarbonyl functional group to achieve FTaseinactivation is described in the examples below, and compounds of thistype offer a new approach to the treatment and control of cancer.Suitable levels of enzyme activity reduction range from at least 10% toat least 95% or even 100%. Any level can be selected as desired, usingappropriate chemical substitutents to achieve at least 10, 20, 30, 40,50, 60, 70, 80, or 90% reduction in enzyme activity. Typical routes ofadministration of these substances that might be suitable for clinicalapplications are oral and intravenous, although other routes might provebeneficial in specific cancers (e.g., intraperitoneal, intramuscular,subcutaneous, intrathecal, or topical for skin cancers). Doses andfrequency of administration are to be selected based upon considerationsof beneficial effects (e.g., tumor growth reduction or tumor shrinkage)compared to side effects (e.g., systemic toxicity). Combination withother anticancer agents to produce synergistic effects of benefit to thepatient are also possible. Combination therapy might be based on twostrategies. One is to interfere with different biochemical processes toincrease tumor cell killing. Another is to hamper development of drugresistance, which is less likely to occur simultaneously in tumor cellsexposed to anticancer agents based on interference with differentbiochemical pathways in the tumor cells.

Compounds according to the invention may be purified free of impurities,preferably to a level of at least 50, 70, 90, or 95%. The compounds maybe formulated in suitable pharmaceutically acceptable diluents,excipients or carriers. These can provide for additional properties,such as enhanced absorption, slow release, tissue or organ targeting,etc.

In summary an improved method of interference with protein prenylationin tumor cells has been described that may prevent or hamper theproliferation of tumor cells, possibly resulting in a decrease in tumorsize and/or disappearance of the cancer.

EXAMPLE 1

An example of an embodiment of this invention is phenylglyoxalmonohydrate, shown below, which is in equilibrium with the dicarbonylform:

The application of phenylglyoxal monohydrate to solutions of FTaseresulted in the rapid and virtually complete inactivation of the enzymein a matter of seconds to minutes, depending upon the concentration ofphenylglyoxal monohydrate applied. For example, the catalytic ability ofFTase was monitored by an assay procedure in which FTase transferred thefarnesyl group from FPP to a peptide substitute for Ras. The peptidesubstitute for Ras in this assay had structural elements in common withRas, in particular a sequence of four amino acidscysteinyl-valyl-lysyl-serine (cys-val-lys-ser). In addition the peptidecontained an amino acid residue that had a fluorescent marker attached,dansyl-glycine (DNS-gly). Farnesyl group transfer by FTase from FPP toDNS-gly-cys-val-lys-ser resulted in an increase in fluorescence due tothe attachment of the hydrophobic group to the cysteine near the dansylgroup. Thus, enzyme activity is revealed by an increase in fluorescenceof the dansyl group, as shown in the accompanying FIG. 1.

Inactivation of the enzyme FTase occurred when the FTase waspreincubated with phenylglyoxal monohydrate, as evidenced by (1) adecrease in enzyme activity with increasing time of preincubation withphenylglyoxal monohydrate; and (2) a decrease in enzyme activity withincreasing concentration of phenylglyoxal monohydrate. Because ofinterference of phenylglyoxal monohydrate with the fluorescence assayused, phenylglyoxal monohydrate was removed from the FTase preincubationmixture by ultrafiltration prior to the assay being performed. Theaccompanying FIG. 2 shows the loss of activity of FTase due toinactivation by phenylglyoxal monohydrate.

Evidence for the binding of phenylglyoxal in the farnesyl pyrophosphatebinding region of the enzyme was obtained by preincubation of the enzymewith farnesyl pyrophosphate, followed by treatment with phenylglyoxal.As shown in the accompanying FIG. 3, higher concentrations of farnesylpyrophosphate more effectively reduced the inactivation of FTase byphenylglyoxal. This suggests that the farnesyl pyrophosphate bound toand blocked phenylglyoxal from the site of the reaction that bringsabout inactivation, namely chemical modification of an active-sitearginine side chain.

A detailed description of the results summarized above follows. Uponfarnesylation by protein farnesyltransferase (FTase),¹ Ras proteinbecomes localized at the inner surface of the cell membrane.Localization is essential for the mitogenic role of mutant Ras protein,which acts as a switch for cancer cell proliferation. We found thatphenylglyoxal (PGO), which is a protein-modification reagent that isspecific for arginine, potently obstructs farnesylation of a Ras modelpeptide by FTase. Covalent modification interferes with catalysis andresults in mechanism-based inactivation of FTase. FTase activity invitro was monitored by a fluorescence assay. FTase was rapidlyinactivated by preincubation with PGO. At 20 mM PGO, FTase activity wastotally lost within 2 minutes. Inactivation rates at 10 mM PGO and 5 mMPGO were successively slower. Preincubation of FTase with FPP prior toincubation with PGO decreased the ability of PGO to inactivate FTase.These results imply that a bimolecular reaction occurred between PGO andFTase, probably in the vicinity of the FPP binding site, which resultedin the inactivation of the enzyme. These findings reveal the value of-dicarbonyl compounds for mechanistic studies of FTase and forpotentially selective regulation of protein farnesylation for possibleanticancer chemotherapy. Footnotes: ¹ The abbreviations used are:Ds-GCVLS, the dansyl-labeled pentapeptideN-dimethylaminonaphthalenesulfonyl-Gly-Cys-Val-Leu-Ser; DTT,dithiothreitol; Fmoc, 9-fluorenylmethoxycarbonyl; FTase, humanrecombinant protein farnesyltransferase; FPP, farnesyl pyrophosphate;GGPP, geranylgeranyl pyrophosphate; GGTase, proteingeranylgeranyltransferase; HEPES,N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; PGO, phenylglyoxalmonohydrate; Tris, tris(hydroxymethyl)aminomethane.

Posttranslational modification of a small number of proteins is carriedout by protein farnesyltransferase (FTase), which attaches the 15-carbonfarnesyl group of farnesyl pyrophosphate to the sulfhydryl group of acysteine residue near the C-terminus of the substrate protein.Noteworthy among the proteins that are substrates for FTase is Rasprotein, which upon farnesylation becomes localized at the lipid bilayerof the inner surface of the cell membrane. Localization is essential forthe mitogenic role of Ras, which acts as a switch for cell division.Cycling between the inactive form (GDP-bound Ras) and the active form(GTP-bound Ras), is part of the normal mechanism of cell growthregulation.

Mutant forms of Ras protein have lost GTPase activity and triggerunrestrained cell division. Approximately one third of all human cancersexhibit a mutant ras genotype, with substantial variations in theincidence from tumor type to tumor type. This has made blockage offarnesylation of mutant Ras protein an active area of research as itappears that inhibition of both normal and mutant Ras proteinfarnesylation is more harmful to cancer cells than normal cells. Thechallenges to this coup de main against cancer, however, are manifold.Some cancer cell lines with mutant ras genotypes are insensitive toFTase inhibitors, and some cancer cell lines without mutant rasgenotypes are sensitive to FTase inhibitors. This may mean that adelicate balance between reduction in farnesylation of differentproteins is required for effective anticancer strategies. A possibleaddition to the armamentarium of farnesylation regulators based on a newapproach is described in this report.

Recognition of Ras and related protein substrates of FTase is based on astructural motif consisting of a C-terminal CAAX sequence, in which Arepresents an aliphatic amino acid and X is preferentially serine. Therelated posttranslational geranylgeranylation, in which the 20-carbongeranylgeranyl chain is attached to the cysteine of CAAX bygeranylgeranyltransferase (GGTase), occurs preferentially when X ismethionine.

Nucleophilic attack by the cysteine sulfhydryl group of CAAX, activatedby an enzyme-bound zinc ion, results in Ras farnesylation, withdeparture of pyrophosphate from FPP. We surmised that catalysis offarnesylation by FTase might be facilitated by salt-bridge formation(i.e., electrostatic stabilization) between the pyrophosphate leavinggroup of farnesyl pyrophosphate and a cationic amino acid side chainsuch as the guanidinium group of arginine. This speculation wasstrengthened by the recent publication of some aspects of FTasestructure obtained from X-ray crystallographic analyses. An arginine(Arg²⁹¹) was found to be in close proximity to the binding site of thediphosphate portion of FPP and the active-site zinc ion, near the upperpart of the barrel-shaped active site. Another key arginine in thebottom of the active site, Arg²⁰², is proposed to form a salt bridgewith the carboxyl group of the CAAX terminus of the Ras peptidesubstrate. In addition the aliphatic part of the arginine side chain isbuttressed with the FPP aliphatic chain. FTase's preference for FPPrelative to GGPP can be understood because the latter would project fromthe cleft and be poorly positioned for transfer of the geranylgeranylgroup to the protein substrates of FTase.

We report herein that a protein-modification agent, phenylglyoxal, thatis specific for the arginine side chain potently obstructs farnesylationof a Ras model peptide by FTase. This approach of specific covalentmodification of FTase to interfere with catalysis results inmechanism-based inactivation of FTase, in contrast to the numerousnoncovalent inhibitors that have been identified. Covalent modificationis in principle virtually irreversible and might be tailored to activesite residues of FTase. Inactivation thus opens the door to mechanisticstudies and potentially selective obstruction of FTase and/or GGTase forpossible anticancer chemotherapeutic benefit.

Experimental Procedures:

Materials. All reagents were from Sigma Chemical Co. and used withoutfurther purification, except as noted.

Purification of PGO—PGO (Aldrich Chemical Co.) was dissolved in boilingwater that contained charcoal, and the mixture was gravity filteredthrough Celite while hot. Upon cooling, the solution was filtered bysuction to obtain the PGO, and the PGO was repeatedly recrystallizedfrom water until its ¹H NMR spectrum and melting point indicated theabsence of impurities, such as polymeric material. All PGO solutionswere freshly prepared before use in the studies described below.Preparation of Ds-GCVLS Ds-GCVLS was prepared by a method analogous to apublished procedure. Synthesis was performed by use of a Millipore 9050Plus PepSynthesizer that employed standard Fmoc solid phase peptidesynthesis methodology. PS-PEG resin and Fmoc protected amino acids wereobtained from Millipore. The N-dansylglycine was from Sigma Chemical Co.Peptide purification was performed by RP-HPLC on an Alltech MacrosphereRP300 C8 column. Elution was accomplished with a linear gradient fromaqueous 10 mM trifluoroacetic acid to 10 mM trifluoroacetic acid inacetonitrile. Lyophilization yielded a pale yellow solid. Stocksolutions were prepared in 50 mM Tris containing 5 mM DTT and 0.2%n-octyl-β-D-glucopyranoside (Anatrace, Inc.), pH 7.7. Concentrations ofDs-GCVLS were determined by use of the extinction coefficient of thedansyl group at 340 nm (=4250 M⁻¹ cm⁻1). Stock solutions were stored at−20 C. FTase (stored at −70 C) was a generous gift from ProfessorPatrick Casey at Duke University.

FTase assay The fluoresence assay conditions were adapted from apublished method. The FTase assay consisted of the measurement of therate of FTase-catalyzed farnesylation of the fluorescent pentapeptideDs-GCVLS. Upon farnesylation of the cysteine, the fluoresence emissionat=505 nm of the dansyl group is enhanced due to the proximity of thehydrophobic isoprenoid group. Enzyme activity (approximately 60 nMFTase) was monitored at saturating substrate concentrations (10 Mfarnesyl pyrophosphate, ammonium salt, prepared by successive dilutionsof a commercial 2.3 mM solution in methanol:10 mM aqueous NH₄OH (7:3)into assay buffer, and 1.0 M Ds-GCVLS) by measurement of the increase influorescence emission observed at=505 nm as FTase farnesylated Ds-GCVLSfor a period of 10 min. The assay buffer was selected for unreactivitytoward PGO: 50 mM NaHEPES, 5 mM DTT, 5 mM MgCl₂, 10 μM ZnCl₂, and 0.2%n-octyl-β-D-glucopyranoside, pH 7.5. Fluorescence emission was measuredwith a JASCO FP-77 spectrofluorimeter fitted with atemperature-controlled cuvette holder connected to a 30 C constanttemperature bath. All fluoresence assays were conducted in a 4 mm 4 mmquartz cuvette with excitation at 340 nm.

Inactivation of FTase by PGO FTase was incubated at 30 C in the presenceand absence of PGO at various concentrations in 50 mM NaHEPES, 5 mMMgCl₂, 10 μM ZnCl₂, and 0.2% n-octyl-β-D-glucopyranoside, pH 7.5 (i.e.,assay buffer without DTT). Periodically, aliquots of the FTase-PGOreaction mixture were removed and assayed for FTase activity. Because ofinterference of PGO with the fluorometric assay, the aliquot was firstsubjected to solvent exchange by size exclusion membrane concentrationto remove PGO. The solvent exchange utilized Microcon (Millipore Co.) 30kD molecular weight cutoff microconcentrators. This also served to stopfurther inactivation of the enzyme by PGO. A 100-L aliquot of FTase-PGOreaction mixture was pipetted into a Microcon concentrator, which wascentrifuged at 12,000 RPM for 8 min at 4 C. The concentrate wasresuspended in 100 L of assay buffer, and centrifugation was repeated.The final concentrate was resuspended in 90 L of assay buffer, and theresulting solution was recovered by inversion of the concentrator andcentrifugation at 3500 RPM for 3 min at 4 C. The solution was thenassayed for FTase activity as described above. A control was handled asabove, except that PGO was omitted from the incubation mixture.

FPP protection of FTase against inactivation by PGO FTase was treatedwith FPP at various concentrations in assay buffer without DTT for 2 minat 30° C. prior to incubation of the mixture with 10 mM PGO for 30 minat 30° C. The control was incubated with FPP at the correspondingconcentration for the full length of time, but without PGO. The mixtureswere subsequently subjected to microconcentration and assay, asdescribed above.

Results and Discussion:

FTase activity in vitro was monitored by a fluorescence assay, shown inFIG. 1, in which activity was determined from the initial slope of aplot of the fluorescence increase vs. time. FTase was rapidlyinactivated by preincubation with PGO, as shown in FIG. 2. At 20 mM PGO,FTase activity was totally lost before the first assay at 2 minutesafter mixing of PGO and FTase (data not shown). Inactivation by 10 mMPGO was slower but still quite rapid. At 5 mM PGO the rate ofinactivation was reduced. These results imply that a bimolecularreaction occurred between PGO and FTase, which resulted in theinactivation of the enzyme.

Inactivation kinetics differ from inhibition kinetics. Inactivationkinetics typically consist of the bimolecular conversion of the activeform of the enzyme (E) into impaired and/or completely inactive form(s)of the enzyme (E′), as shown in the equation below, where PGO is theinactivator:

Because the inactivator is typically present in large excess over theenzyme and therefore remains at essentially constant concentration,disappearance of enzyme activity follows pseudo-first-order kinetics,described by the following equation:[E] _(t) /[E] ₀=exp(−k[PGO]t)in which [E]_(t) is the enzyme concentration or activity remaining attime t, [E]₀ is the initial enzyme concentration or activity, and [PGO]is the concentration of PGO. Thus, a plot of log [E]_(t)/[E]₀, i.e., log(fraction of activity remaining), versus time is expected to be linear,with a slope of k[PGO]/2.303. Thus, the time-dependence andinhibitor-concentration-dependence of loss of enzyme activity areindicative of inactivation of the enzyme.

Previously, the stepwise formation of a 2:1 PGO-enzyme adduct has beenseen kinetically as a biphasic time course of inactivation of porcineliver prenyltransferase, presumably due to rapid, reversible 1:1 adductformation resulting in reduced activity of the enzyme, followed byslower, irreversible 2:1 adduct formation, and conversion of the enzymeinto a less active form. Some indication of similar behavior is seen inFIG. 2, especially at low PGO concentration.

Preincubation of FTase with FPP prior to incubation with PGO decreasedthe ability of PGO to inactivate FTase, as shown in FIG. 3. This impliesthat an active site arginine residue is the principal site of thereaction with PGO that inactivates the enzyme. When argininemodification occurs in the FPP binding site, then obstruction ofsubsequent binding of FPP would be expected to prevent farnesylation ofthe substrate pentapeptide. The low concentration of PGO required forinactivation of the enzyme is also consistent with the idea thatspecific modification of an active-site arginine has occurred.

It is not possible to rule out that the arginine side-chain modificationresults in destruction of the catalytic ability of the active-sitearginine without blockage of FPP binding. Likewise, some modification oflysine side chains and -amino groups is possible with phenylglyoxal,although under the short incubation times used in these studies, suchmodification would be expected to be of minor importance. Destruction ofenzyme activity by modification of numerous arginines, which might forexample lead to gross conformational changes and concomitant loss ofcatalytic ability, also would not be expected to occur under theseconditions. In either case, the observed protective effect of FPP wouldnot necessarily have been expected. Future studies, however, arerequired to establish whether Arg²⁹¹ is the principal site ofmodification of FTase by PGO. Such studies will also ascertain whetheranother amino acid is a significant target of PGO modification.

EXAMPLE 2

In the same manner as Example 1, compound 2-oxododecanal was tested forinactivation of FTase in the fluorescence assay system described, exceptas noted below. Final concentrations in the incubation mixture were 5,10, and 35 mM 2-oxododecanal. A stock solution of 2-oxododecanaldissolved in dimethyl sulfoxide (DMSO) was employed. The finalconcentration of DMSO in the incubation mixture (as well as the control)was 5%. The results are shown in FIG. 4. It was found that2-oxododecanal rapidly inactivated FTase, probably by reaction with oneor both of the active site arginine residues. This would block access ofone or both of the substrates (FPP and/or the peptide substitute for Rasprotein) and/or block the catalytic function of one or both of thearginine residues, resulting in inactivation of the enzyme.

EXAMPLE 3

In the same manner as EXAMPLE 1, compound5,9-dimethyl-8-decene-2,3-dione was tested for inactivation of FTase inthe fluorescence assay system described, except as noted below. A stocksolution of 5,9-dimethyl-8-decene-2,3-dione in 5% DMSO was employed. Thefinal concentration of DMSO in the incubation mixture (as well as thecontrol) was 4.6%. FTase was at 15 nM, and the Microcon concentrationstep was omitted. It was found that 5,9-dimethyl-8-decene-2,3-dionerapidly inactivated FTase. After 30 minutes of incubation with 17, 68,or 171 μM 5,9-dimethyl-8-decene-2,3-dione, FTase activity was reduced to38%, 18 or 16% of the initial control value, respectively.

EXAMPLE 4

In the same manner as EXAMPLE 1, dehydroascorbic acid 6-palmitate wastested for inactivation of FTase in the fluorescence assay systemdescribed, except as noted below. A stock solution of dehydroascorbicacid 6-palmitate in 2% DMSO in 5 mM sodium acetate, pH 3.9, wasemployed. The final concentration of DMSO in the incubation mixture (aswell as the control) was 0.46%. FTase was at 15 nM, and the Microconconcentration step was omitted. It was found that dehydroascorbic acid6-palmitate rapidly inactivated FTase. After 30 minutes of incubationwith 7, 28, or 141 μM dehydroascorbic acid 6-palmitate, FTase activitywas reduced to 75%, 34%, or 22% of the initial control value,respectively.

EXAMPLE 5

In the same manner as EXAMPLE 1, compound 4,4′-biphenyldiglyoxaldehydewas tested for inactivation of FTase in the fluorescence assay systemdescribed, except as noted below. A stock solution of4,4′-biphenyldiglyoxaldehyde in 5% DMSO was employed. The finalconcentration of DMSO in the incubation mixture (as well as the control)was 4.6%, and FTase was at 15 nM. It was found that4,4′-biphenyldiglyoxaldehyde rapidly inactivated FTase. After 30 minutesof incubation with 23 or 93 μM 4,4′-biphenyldiglyoxaldehyde, FTaseactivity was reduced to 68% or 11% of the initial control value,respectively.

EXAMPLE 6

As a test of the importance of the presence of a dicarbonyl functionalgroup, a compound analogous to that used in EXAMPLE 5 but without such agroup was tested. Thus, in the same manner as EXAMPLE 1, compound4,4′-diacetylbiphenyl, whose structure is as follows:

was tested for inactivation of FTase in the fluorescence assay systemdescribed, except as noted below. A stock solution of4,4′-diacetylbiphenyl in 5% DMSO was employed. The final concentrationof DMSO in the incubation mixture (as well as the control) was 4.6%,FTase was at 15 nM. It was found that 4,4′-diacetylbiphenyl did notinactivated FTase. Even after 120 minutes of incubation with 23 μM4,4′-diacetylbiphenyl, FTase activity was the same as the control,within experimental error.

EXAMPLE 7

In the same manner as EXAMPLE 1, compound 2,3-pentanedione,CH₃CH₂(C═O)—(C═O)CH₃, was tested for inactivation of FTase in thefluorescence assay system described, except as noted below. The assaywas carried out in the presence of 50 mM sodium borate, pH 7.5. FTasewas at 15 nM, and the Microcon concentration step was omitted. It wasfound that 2,3-pentanedione rapidly inactivated FTase. Afterapproximately 30 minutes of incubation with 17, 33, or 66 mM2,3-pentanedione, FTase activity was reduced to 69%, 55%, or 37% of theinitial control value, respectively.

References

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1. A compound of Formula IR1-(C═O)—(C═O)—R2 wherein R1 is selected from the group consisting of

or R1 is a farnesyl-mimicking end or a geranylgeranyl-mimicking end ofthe formula

wherein n is from 0 to 10, and wherein R2 is H, alkyl, or alkenyl, withthe proviso that R1 and R2 are not both 2,6 dimethyl-5-heptenyl, wherebysaid compound is not2,6,11,15-tetramethyl-hexadeca-2,14-diene-8,9-dione.
 2. A compound ofclaim 1 wherein R2 is alkenyl having from two to fifteen carbon atoms.3. A compound of claim 1 which is selected from the group consisting of3,7-dimethyl-2-oxo-6-octenal, 3,7,11-trimethyl-2-oxododeca-6,10-dienal,7,11-dimethyldodeca-6,10-dien-2,3-dione,7,11,15-trimethylhexadeca-6,10,14-trien-2,3-dione,5,9-dimethyl-8-decene-2,3-dione, and6,10,14-trimethyl-2-oxopentadeca-5,9,13 -trienal.
 4. A compound of claim1 wherein R2 is H or linear alkyl having from 1 to 25 carbon atoms.
 5. Acompound of claim 3 which is 5,9-dimethyl-8-decene-2,3-dione.
 6. Amethod of inactivating a prenylation enzyme comprising contacting aprenylation enzyme with a compound of claim
 1. 7. A method of screeningcompounds as anti-tumor agents comprising contacting a test compound ofclaim 1 with a prenylation enzyme, wherein a test compound which reducesprenylation activity at least 50% is a candidate anti-tumor agent.
 8. Amethod of inhibiting the growth of a cancer cell comprising contacting acancer cell with a compound of claim 1, whereby the growth of the cancercell is inhibited.
 9. A pharmaceutical formulation comprising a compoundof claim 1 and a pharmaceutically acceptable excipient.
 10. The compoundof claim 4 wherein R2 is H or methyl.